Novel cytochrome p450 enzymes from sorghum bicolor

ABSTRACT

Two novel cytochrome P450 genes are isolated from sorghum, each gene encoding a protein having pentadecatrienyl resorcinol hydroxylase activity. Expression vectors containing these sequences are made and used to elevate levels of pentadecatrienyl resorcinol hydroxylase in transgenic cells and organisms.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to two novel pentadecatrienyl resorcinolhydroxylases which are in the cytochrome P450 gene superfamily. Thisinvention relates to the DNA sequences of these novel enzymes, the aminoacid sequences of these novel enzymes, expression vectors containing theDNA sequences of these novel enzymes, methods of making these novelenzymes, methods of transforming plants to express these novel enzymes,and transgenic plants expressing these novel enzymes which may result inthe production of dihydrosorgoleone which may be secreted by a plant andundergo oxidation to become sorgoleone.

2. Prior Art Description

Allelopathy, sometimes referred to as a form of chemical warfare betweenplants, can be defined as the production and release of chemicalsubstances by one species that inhibit the growth of another species(Inderjit and Duke, Planta 217:529-539 (2003); Weston and Duke, Crit.Rev. Plant Sci. 22:367-389 (2003)). Allelopathic interactions have beenproposed to have profound effects on the evolution of plant communitiesthrough the loss of susceptible species via chemical interference, andby imposing selective pressure favoring individuals resistant toinhibition from a given allelochemical (e.g., Schulz and Wieland.Chemoecology 9:133-141 (1999)). Furthermore, allelopathic compoundsreleased by grain crop species are thought to play a significant role incover crops or within intercropping systems where they act as weedsuppressants. Allelopathic compounds have been characterized in a numberof plants such as black walnut, wheat, rice, and sorghum (Bertin, etal., Plant and Soil 256: 67-83 (2003); Inderjit and Duke, (2003); Dukeet al., Outlooks Pest Management 16: 64-68 (2005)). These chemicalswhich can be produced and released by many types of plants, algae,bacteria, and fungi and which often involve secondary metabolites, arereferred to as allelochemicals or phytotoxins when produced by plants.Of note, allelopathy and allelochemicals can also positively influencethe growth, survival, and reproduction of other organisms.

Despite the ecological and agronomic importance of allelochemicals,relatively few pathways have been characterized in detail at themolecular level. One notable exception is the identification andcharacterization of all the genes encoding the enzymes responsible forthe biosynthesis of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one,a benzoxazinoid, in Zea mays (Frey et al., Science 277:696-699 (1997)).Benzoxazinoids are thought to act as alleopathic chemicals in therhizosphere, in addition to being defense compounds against microbialpathogens and insect herbivores (Sicker et al., Int. Rev. Cytol.198:319-346 (2000): Friebe, J. Crop Prod. 4:379-400 (2001)).

Several Sorghum species have been reported to produce phytotoxins(secondary metabolites) which are exuded from their root systems intothe rhizosphere, which suppress the growth of competing species(Einhellig, Agronomy Journal 88:886-893 (1996)). Numerous studies havecontributed to the discovery and identification of the chemicals thatare responsible for this observed allelopathic inhibition. For example,studies on the biologically-active components of both water-soluble andwater-insoluble exudates from roots of Sorghum bicolor have demonstratedtheir role in the growth inhibition of lettuce seedlings (Lactucasativa), as well as a number of important invasive weed species (Netzlyand Butler, Crop Science 26:775-778 (1986)). The major constituent ofthese exudates was identified as2-hydroxy-5-methoxy-3-[(Z,Z)-8′,11′,14′-pentadecatriene]-p-benzoquinone,referred to as sorgoleone (Chang, et al., J. Am. Chem. Soc.108:7858-7860 (1986)), which has been estimated to account for as muchas 85% of the exudate material (w/w) in some varieties of sorghum(Czarnota, et al., Weed Technology 15:815-825 (2001)). The remainingexudate consists largely of sorgoleone congeners differing in the lengthor degree of saturation of the aliphatic side chain, and in thesubstitution pattern of the quinone ring (Kagan, et al., J. Agric. FoodChem. 51:7589-7595 (2003): Rimando, et al., J. Nat. Prod. 66:42-45(2003)). Sorgoleone acts as a potent broad-spectrum inhibitor activeagainst many agronomically important monocot and dicot weed species,exhibits a long half-life in soil, and appears to affect multipletargets in vivo (e.g., Netzly and Butler, 1986; Einhellig and Souza. J.Chemical Ecology 18:1-11 (1992); Nimbal, et al., J. Agric. Food Chem.44:1343-1347 (1996); Czarnota, et al., 2001; Bertin, et al., 2003; Duke,Trends Biotechnol. 21:192-195 (2003)). Sorgoleone is a promising naturalproduct alternative to synthetic herbicides (Duke, 2003).

The herbicidal and allelopathic properties of sorgoleone make theisolation and characterization of the corresponding genes involved insorgoleone biosynthesis highly desirable, as manipulation of the pathwayin sorghum, or genetic modification of other plant species using thesegenes could provide important insights into the underlyingallelochemical interactions involved (Duke, 2003). Sorgoleonebiosynthesis is likely restricted to root hairs, which appear ascytoplasmically dense cells in sorghum, containing large osmiophilicglobules deposited between the plasmalemma and cell wall, presumablyassociated with sorgoleone rhizosecretion (Czarnota, et al. Int. J.Plant Sci. 164:861-866 (2003b); Czarnota, et al., J. Chem. Ecology29:2073-2083 (2003a)). The biosynthetic pathway of sorgoleone appears torequire four types of enzymes: fatty acid desaturases, polyketidesynthases, O-methyltransferases, and cytochrome P450 monooxygenases(FIG. 1). Recently, the following enzymes in this pathway have beencharacterized: two S. bicolor fatty acid desaturases (DES2, DES3; Pan,et al., J. Biol. Chem. 282:4326-4335 (2007) and U.S. Pat. No.8,383,890), two alkylresorcinol synthases (ARS1, ARS2; U.S. Patent Pub.2011-0225676), and a 5-n-alk(en)ylresorcinol-utilizingO-methyltransferase (OMT3; U.S. Pat. No. 7,732,666) which likelyparticipate in the biosynthesis of sorgoleone. As of yet, the enzyme orenzymes that convert 3-methyl-5-pentadecatrienyl resorcinol todihydrosorgoleone, a vital step in the biosynthesis of sorgoleone, havenot been isolated or characterized.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of this invention to have two novel pentadecatrienylresorcinol hydroxylases with distinct DNA sequences.

It is an object of this invention to have an isolated polynucleotideencoding each novel pentadecatrienyl resorcinol hydroxylase. It is afurther object of this invention that the amino acid sequence ofpentadecatrienyl resorcinol hydroxylase is SEQ ID NO: 12 and SEQ ID NO:14. It is another object of this invention to have an isolatedpolynucleotide encoding a polypeptide that is at least 99%, at least98%, at least 97%, at least 96%, at least 95%, at least 94%, at least93%, at least 92%, at least 91%, at least 90%, at least 89%, at least88%, at least 87%, at least 86%, at least 85% identical to each novelpentadecatrienyl resorcinol hydroxylase.

It is an object of this invention to have an isolated polynucleotideencoding one of the novel pentadecatrienyl resorcinol hydroxylases or apolypeptide that is at least 95%, at least 90% or at least 85% identicalto the amino acid sequence of one of the novel pentadecatrienylresorcinol hydroxylases. It is a further object of this invention tohave a promoter operatively linked to the polynucleotide encoding one ofthe novel pentadecatrienyl resorcinol hydroxylases or encoding apolypeptide that is at least 95%, at least 90% or at least 85% identicalto the amino acid sequence of one of the novel pentadecatrienylresorcinol hydroxylases. It is another object on this invention that thepromoter is active in one or more of the following: plants, fungi,blue-green algae, bacteria, and animals.

It is an object of this invention to have an isolated polynucleotideencoding one of the novel pentadecatrienyl resorcinol hydroxylases or apolypeptide that is at least 95%, at least 90% or at least 85% identicalto the amino acid sequence of one of the novel pentadecatrienylresorcinol hydroxylases. It is a further object of this invention tohave a promoter operatively linked to the polynucleotide encoding one ofthe novel pentadecatrienyl resorcinol hydroxylases or encoding apolypeptide that is at least 95%, at least 90% or at least 85% identicalto the amino acid sequence of one of the novel pentadecatrienylresorcinol hydroxylases. It is another object on this invention that thepromoter is active in one or more of the following: plants, fungi,blue-green algae, bacteria, and animals. It is a further object of thisinvention that the promoter be either an inducible promoter or aconstitutive promoter. It is another object of this invention that thepromoter be a tissue-specific promoter.

It is an object of this invention to have an isolated polynucleotideencoding each novel pentadecatrienyl resorcinol hydroxylase or apolypeptide that is at least 95%, at least 90% or at least 85% identicalto the amino acid sequence of each novel pentadecatrienyl resorcinolhydroxylase. It is a further object of this invention to have a promoteroperatively linked to the polynucleotide encoding each novelpentadecatrienyl resorcinol hydroxylase or encoding a polypeptide thatis at least 95%, at least 90% or at least 85% identical to the aminoacid sequence of each novel pentadecatrienyl resorcinol hydroxylase. Itis another object on this invention that the promoter is active inplants, and more specifically in the root cells of a plant, and morespecifically in the root hair cells of a plant.

It is an object of this invention to have an expression vectorcontaining a promoter operatively linked to a polynucleotide encodingone of the following: one of the novel pentadecatrienyl resorcinolhydroxylases, or a polypeptide that is at least 95%, at least 90% or atleast 85% identical to the amino acid sequence of one of the novelpentadecatrienyl resorcinol hydroxylases. It is another object on thisinvention that the promoter is active in one or more of the following:plants, fungi, blue-green algae, bacteria, and animals. It is a furtherobject of this invention that the promoter be either an induciblepromoter or a constitutive promoter. It is another object of thisinvention that the promoter be a tissue-specific promoter.

It is an object of this invention to have a transformed cell which istransformed with an expression vector containing a promoter operativelylinked to a polynucleotide encoding one of the following: one of thenovel pentadecatrienyl resorcinol hydroxylases, or a polypeptide that isat least 95%, at least 90% or at least 85% identical to the amino acidsequence of one of the novel pentadecatrienyl resorcinol hydroxylases.It is another object of this invention that the promoter is active inone or more of the following: plants, fungi, blue-green algae, bacteria,and animals. It is a further object of this invention that the promoterbe either an inducible promoter or a constitutive promoter. It isanother object of this invention that the promoter be a tissue-specificpromoter.

It is an object of this invention to have a transformed cell which istransformed with an expression vector containing a promoter operativelylinked to a polynucleotide encoding one of the following: one of thenovel pentadecatrienyl resorcinol hydroxylases, or a polypeptide that isat least 95%, at least 90% or at least 85% identical to the amino acidsequence of one of the novel pentadecatrienyl resorcinol hydroxylases.It is another object of this invention that the promoter is active inone or more of the following: plants, fungi, blue-green algae, bacteria,and animals. It is a further object of this invention that the promoterbe either an inducible promoter or a constitutive promoter ortissue-specific promoter. It is a further object of this invention thatthe transformed cell be a plant cell, a fungus, a blue-green algae, or abacterium.

It is an object of this invention to have a transformed cell which istransformed with an expression vector containing a promoter operativelylinked to a polynucleotide encoding one of the following: one of thenovel pentadecatrienyl resorcinol hydroxylases, or a polypeptide that isat least 95%, at least 90% or at least 85% identical to the amino acidsequence of one of the novel pentadecatrienyl resorcinol hydroxylases.It is another object of this invention that the promoter is active inplants, and more specifically in the root cells of a plant, and morespecifically in the root hair cells of a plant. It is another object ofthis invention that the transformed cell be a plant cell.

It is an object of this invention to have a transformed organismcontains a polynucleotide encoding one of the novel pentadecatrienylresorcinol hydroxylase or a polypeptide that is at least 95%, at least90% or at least 85% identical to the amino acid sequence of one of thenovel pentadecatrienyl resorcinol hydroxylases. It is another object ofthis invention that the transformed organism has elevated levels ofpentadecatrienyl resorcinol hydroxylase compared to a wild-typeorganism. It is another object of this invention that the organism canbe a plant, fungi, blue-green algae, or bacteria. It is further objectof this invention that the polynucleotide be operably linked to apromoter that is active in the organism. It is another object of thisinvention that the promoter be an inducible promoter, a constitutivepromoter, or a tissue-specific promoter.

It is an object of this invention to have an isolated polynucleotideencoding each novel pentadecatrienyl resorcinol hydroxylase. It is afurther object of this invention that the DNA sequence ofpentadecatrienyl resorcinol hydroxylase is SEQ ID NO: 11 and SEQ ID NO:13. It is another object of this invention to have an isolatedpolynucleotide having a sequence that is at least 99%, at least 98%, atleast 97%, at least 96%, at least 95%, at least 94%, at least 93%, atleast 92%, at least 91%, at least 90%, at least 89%, at least 88%, atleast 87%, at least 86%, at least 85% identical to the DNA sequence ofeach novel pentadecatrienyl resorcinol hydroxylase.

It is an object of this invention to have an isolated polynucleotide forone of the novel pentadecatrienyl resorcinol hydroxylase genes or a DNAsequence that is at least 95%, at least 90% or at least 85% identical tothe DNA sequence of one of the novel pentadecatrienyl resorcinolhydroxylase genes. It is a further object of this invention to have apromoter operatively linked to the polynucleotide for one of the novelpentadecatrienyl resorcinol hydroxylases or a DNA sequence that is atleast 95%, at least 90% or at least 85% identical to the DNA sequence ofone of the novel pentadecatrienyl resorcinol hydroxylase genes. It isanother object on this invention that the promoter is active in one ormore of the following: plants, fungi, blue-green algae, bacteria, andanimals.

It is an object of this invention to have an isolated polynucleotide forone of the novel pentadecatrienyl resorcinol hydroxylase genes or a DNAsequence that is at least 95%, at least 90% or at least 85% identical tothe DNA sequence of one of the novel pentadecatrienyl resorcinolhydroxylase genes. It is a further object of this invention to have apromoter operatively linked to the polynucleotide for one of the novelpentadecatrienyl resorcinol hydroxylase genes or a DNA sequence that isat least 95%, at least 90% or at least 85% identical to the DNA sequenceof one of the novel pentadecatrienyl resorcinol hydroxylase genes. It isanother object on this invention that the promoter is active in one ormore of the following: plants, fungi, blue-green algae, bacteria, andanimals. It is a further object of this invention that the promoter bean inducible promoter, a constitutive promoter, or a tissue-specificpromoter.

It is an object of this invention to have an isolated polynucleotide forone of the novel pentadecatrienyl resorcinol hydroxylase genes or a DNAsequence that is at least 95%, at least 90% or at least 85% identical tothe DNA sequence of one of the novel pentadecatrienyl resorcinolhydroxylase genes. It is a further object of this invention to have apromoter operatively linked to the polynucleotide for one of the novelpentadecatrienyl resorcinol hydroxylase genes or a DNA sequence that isat least 95%, at least 90% or at least 85% identical to the DNA sequenceof one of the novel pentadecatrienyl resorcinol hydroxylase genes. It isanother object on this invention that the promoter is active in plants,and more specifically in the root cells of a plant, and morespecifically in the root hair cells of a plant.

It is an object of this invention to have an expression vectorcontaining a promoter operatively linked to a polynucleotide having oneof the following DNA sequences: one of the novel pentadecatrienylresorcinol hydroxylase genes, or a DNA sequence that is at least 95%, atleast 90% or at least 85% identical to the DNA sequence of one of thenovel pentadecatrienyl resorcinol hydroxylase genes. It is anotherobject on this invention that the promoter is active in one or more ofthe following: plants, fungi, blue-green algae, bacteria, and animals.It is a further object of this invention that the promoter be aninducible promoter, a constitutive promoter, or a tissue specificpromoter.

It is an object of this invention to have transformed cell which istransformed with an expression vector containing a promoter operativelylinked to a polynucleotide having one of the following DNA sequences:one of the novel pentadecatrienyl resorcinol hydroxylase genes, or a DNAsequence that is at least 95%, at least 90% or at least 85% identical tothe DNA sequence of one of the novel pentadecatrienyl resorcinolhydroxylase genes. It is another object on this invention that thepromoter is active in one or more of the following: plants, fungi,blue-green algae, bacteria, and animals. It is a further object of thisinvention that the promoter be an inducible promoter, a constitutivepromoter, or a tissue-specific promoter.

It is an object of this invention to have transformed cell which istransformed with an expression vector containing a promoter operativelylinked to a polynucleotide having one of the following DNA sequences:one of the novel pentadecatrienyl resorcinol hydroxylase genes, or a DNAsequence that is at least 95%, at least 90% or at least 85% identical tothe DNA sequence of one of the novel pentadecatrienyl resorcinolhydroxylase genes. It is another object on this invention that thepromoter is active in one or more of the following: plants, fungi,blue-green algae, bacteria, and animals. It is a further object of thisinvention that the promoter be an inducible promoter, a constitutivepromoter, or a tissue-specific promoter. It is a further object of thisinvention that the transformed cell be a plant cell, a fungus, ablue-green algae, or a bacterium.

It is an object of this invention to have transformed cell which istransformed with an expression vector containing a promoter operativelylinked to a polynucleotide having one of the following DNA sequences:one of the novel pentadecatrienyl resorcinol hydroxylase genes, or a DNAsequence that is at least 95%, at least 90% or at least 85% identical tothe DNA sequence of one of the novel pentadecatrienyl resorcinolhydroxylase genes. It is another object on this invention that thepromoter is active in plants, and more specifically in the root cells ofa plant, and more specifically in the root hair cells of a plant. It isanother object of this invention that the transformed cell be a plantcell.

It is an object of this invention to have a transformed organismcontains a polynucleotide having a DNA sequence of one of the novelpentadecatrienyl resorcinol hydroxylase genes or a DNA sequence that isat least 95%, at least 90% or at least 85% identical to the DNA sequenceof one of the novel pentadecatrienyl resorcinol hydroxylase genes. It isanother object of this invention that the transformed organism haselevated levels of pentadecatrienyl resorcinol hydroxylase compared to awild-type organism. It is another object of this invention that theorganism can be a plant, fungi, blue-green algae, or bacteria. It isfurther object of this invention that the polynucleotide be operablylinked to a promoter that is active in the organism.

It is an object of this invention to have a method to producedihydrosorgoleone in a plant or a plant cell. It is a further object ofthis invention to have a method of producing dihydrosorgoleone in aplant or a plant cell by transforming the plant or plant cell withpolynucleotides encoding two fatty acid desaturases, an alkylresorcinolsynthase, an O-methyltransferase, and a pentadecatrienyl resorcinolhydroxylase and allowing the polynucleotides to be expressed in theplant or plant cell. It is another object of this invention that thepolynucleotides are contained in one or more expression vectors. It isstill another object of this invention that each of the polynucleotidesare operably linked to a promoter and further that the promoter isactive in the plant or plant cell.

It is an object of this invention to have a method to producedihydrosorgoleone in a plant or a plant cell. It is a further object ofthis invention to have a method of producing dihydrosorgoleone in aplant or a plant cell by transforming the plant or plant cell withpolynucleotides encoding two fatty acid desaturases, an alkylresorcinolsynthase, an O-methyltransferase, and a pentadecatrienyl resorcinolhydroxylase and allowing the polynucleotides to be expressed in theplant or plant cell. It is another object of this invention that thepolynucleotides are contained in one or more expression vectors. It isstill another object of this invention that each of the polynucleotidesare operably linked to a promoter and further that the promoter isactive in the plant or plant cell. It is a further object of thisinvention that the two fatty acid desaturases are SbDES2 and SbDES3, thealkylresorcinol synthase is either SbARS1 or SbARS2, theO-methyltransferase is SbOMT3, and the pentadecatrienyl resorcinolhydroxylase is either SbPRH1 or SbPRH2.

It is another object of this invention to have a transformed cell havingelevated levels of pentadecatrienyl resorcinol hydroxylase compared tountransformed cells. It is a further object of this invention that thetransformed cells contain an expression vector which encodespentadecatrienyl resorcinol hydroxylase.

It is an object of this invention to have an isolated polypeptide whichhas one of the following amino acid sequences: the amino acid sequenceof SEQ ID NO: 12; an amino acid sequence that is at least 99%, at least98%, at least 97%, at least 96%, at least 95%, at least 94%, at least93%, at least 92%, at least 91%, at least 90%, at least 89%, at least88%, at least 87%, at least 86%, or at least 85% identical to the aminoacid sequence of SEQ ID NO: 12: the amino acid sequence of SEQ ID NO:14; or an amino acid sequence that is at least 99%, at least 98%, atleast 97%, at least 96%, at least 95%, at least 94%, at least 93%, atleast 92%, at least 91%, at least 90%, at least 89%, at least 88%, atleast 87%, at least 86%, or at least 85% identical to the amino acidsequence of SEQ ID NO: 14.

It is another object of this invention to have a method of manipulatingpentadecatrienyl resorcinol hydroxylase levels in a cell or organism. Itis another object of this invention to have a method of manipulatingpentadecatrienyl resorcinol hydroxylase levels in a cell or organism byintroducing into the cell or organism an expression vector containing apolynucleotide operably linked to a promoter and allowing the productionof pentadecatrienyl resorcinol hydroxylase in the cell or organism. Itis a further object of this invention that the polynucleotide encodes apolypeptide having one of the following amino acid sequences: the aminoacid sequence of SEQ ID NO: 12; an amino acid sequence that is at least99%, at least 98%, at least 97%, at least 96%, at least 95%, at least94%, at least 93%, at least 92%, at least 91%, at least 90%, at least89%, at least 88%, at least 87%, at least 86%, or at least 85% identicalto the amino acid sequence of SEQ ID NO: 12; the amino acid sequence ofSEQ ID NO: 14; or an amino acid sequence that is at least 99%, at least98%, at least 97%, at least 96%, at least 95%, at least 94%, at least93%, at least 92%, at least 91%, at least 90%, at least 89%, at least88%, at least 87%, at least 86%, or at least 85% identical to the aminoacid sequence of SEQ ID NO: 14. It is yet a further object of thisinvention that the promoter is active in the cell or organism. It isanother object of this invention that the promoter is an induciblepromoter, a constitutive promoter, or a tissue-specific promoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the proposed biosynthetic pathway of sorgoleone.

FIG. 2 shows the levels of expression of SbPRH1 and SbPRH2 in variousplant tissues as determined by qRT-PCR.

FIGS. 3A, 3B, 3C, and 3D show the extracted ion chromatogram (EIC) forthe ion m/z 400 and 576 of dihydrosorgoleone (FIG. 3A), yeasttransformed with CYP71AM1 (FIG. 3B) or CYP71V7 (FIG. 3C), and negativecontrol yeast transformed with empty vector (FIG. 3D), demonstratingthat the yeast transformed with CYP71AM1 or with CYP71V7 convert3-methyl-5-pentadecatrienyl resorcinol to dihydrosorgoleone. The EIC isshown detailing a single intense peak at 13.03 min for TMS derivatizedsubstrate (m/z=400) and 14.16 min for dihydrosorgoleone (m/z=576).

FIGS. 4A, 4B, and 4C are the mass spectra of TMS-derivatized products.FIG. 4A is the mass spectrum for sorgoleone extracted from sorghumroots. FIGS. 4B and 4C are the mass spectra for extracts from yeastcells expressing CYP71AM1 and CYP71V7, respectively, exogenouslyprovided with 3-methyl-5-pentadecatrienyl resorcinol via the culturemedia.

DETAILED DESCRIPTION OF THE INVENTION

This invention involves the isolation, sequencing, and functionalcharacterization of two cytochrome P450 monooxygenases, designatedCYP7V7 and CYP71AM1, which convert 5-pentadecatrienylresorcinol-3-methyl ester to dihydrosorgoleone, a reduced form ofsorgoleone, which, upon rhizosecretion, rapidly undergoes oxidation tothe benzoquinone sorgoleone (see, Cook, et al., Plant Cell 22:867-887(2010); Dayan, et al., Phytochemistry 71:1032-1039 (2010); FIG. 1). Theisolation, sequencing, and characterization of CYP71AM1 and CYP71V7 fromsorghum provides new genetic engineering opportunities in plants, notonly for altering sorgoleone content leading to the generation of novelgermplasm possessing enhanced agronomic characteristics, but also forthe use of plants cells as bioreactors, thus providing an efficientsource for obtaining sorgoleone and related phenolic lipids inlarge-scale. In addition to plants transformed with CYP71V7 and/orCYP71AM1 and producing the gene product(s), one can transform fungi orgreen algae with one or both of these genes. Then the transformed fungior transformed green algae can produce the gene product(s) and convert5-pentadecatrienyl resorcinol-3-methyl ester to dihydrosorgoleone. Alsothe transformed fungi or green algae can generate certain phenoliclipids by producing one or both of these genes and providing specificsubstrates to the transformed green algae and/or fungi.

The terms “isolated”. “purified”, or “biologically pure” as used herein,refer to material that is substantially or essentially free fromcomponents that normally accompany the material in its native state. Inan exemplary embodiment, purity and homogeneity are determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A nucleicacid that is the predominant species present in a preparation issubstantially purified. In an exemplary embodiment, the term “purified”denotes that a nucleic acid or protein gives rise to essentially oneband in an electrophoretic gel. Typically, isolated nucleic acids orproteins have a level of purity expressed as a range. The lower end ofthe range of purity for the component is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

The term “nucleic acid” as used herein, refers to a polymer ofribonucleotides or deoxyribonucleotides. Typically, “nucleic acid”polymers occur in either single- or double-stranded form, but are alsoknown to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers aswell as nucleic acids comprising known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Exemplary analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs). “DNA”, “RNA”, “polynucleotides”, “polynucleotidesequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleicacid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and“isolated nucleic acid fragment” are used interchangeably herein.

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). Estimates are typically derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (see e.g., Batzer et al.,Nucleic Acid Res. 19:5081 (1991): Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes8:91-98(1994)).

In addition to the degenerate nature of the nucleotide codons whichencode amino acids, alterations in a polynucleotide that result in theproduction of a chemically equivalent amino acid at a given site, but donot affect the functional properties of the encoded polypeptide, arewell known in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine or histidine,can also be expected to produce a functionally equivalent protein orpolypeptide.

As used herein a nucleic acid “probe”, oligonucleotide “probe”, orsimply a “probe” refers to a nucleic acid capable of binding to a targetnucleic acid of complementary sequence through one or more types ofchemical bonds, usually through complementary base pairing, usuallythrough hydrogen bond formation. As used herein, a probe may includenatural (i.e., A, G, C, or T) or modified bases (e.g., 7-deazaguanosine,inosine, etc.). In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. Probes may contain a label so that one candetermine if the probe is bound to the target sequence. By assaying forthe presence or absence of the probe, one can detect the presence orabsence of the select sequence or subsequence.

The term “label” as used herein, refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. A probe can be bound, either covalently, through a linker or achemical bond, or noncovalently, through ionic, van der Waals,electrostatic, or hydrogen bonds, to a label such that the presence ofthe probe may be detected by detecting the presence of the label boundto the probe. Exemplary labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available. In one exemplary embodiment, labelscan be isotopes, chromophores, lumiphores, chromogens, etc. Labels canalso involve two or more compounds, only one of which need be attachedto the probe. An example of a pair of compounds that are labels isbiotin and streptavidin, where biotin is attached to the probe and laterreacts with streptavidin which is added after the probe binds the targetsequence. In this embodiments, the probes are indirectly labeled,because the label (streptavidin) later binds to the biotin which isattached to the probe.

The term “primer” as used herein, refers to short nucleic acids,typically a DNA oligonucleotide of at least about 15 nucleotides inlength. In an exemplary embodiment, primers are annealed to acomplementary target DNA or RNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA or RNA strand.Annealed primers are then extended along the target strand by a DNApolymerase enzyme or reverse transcriptase. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart. PCR primer pairs are typically derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5 © 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

One of ordinary skill in the art will appreciate that the specificity ofa particular probe or primer increases with its length. Thus, forexample, a primer or probe comprising 20 consecutive nucleotides of aparticular gene sequence will anneal to the target sequence with ahigher specificity than a corresponding primer or probe of only 15nucleotides. Thus, in an exemplary embodiment, greater specificity of anucleic acid primer or probe is attained with probes and primersselected to comprise 20, 25, 30, 35, 40, 50 or more consecutivenucleotides of a selected sequence.

Nucleic acid probes and primers are readily prepared based on thenucleic acid sequences disclosed herein. Methods for preparing and usingprobes and primers and for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, e.g., in Green andSambrook, Molecular Cloning, A Laboratory Manual 4th ed. 2012, ColdSpring Harbor Laboratory: and Ausubel et al., eds., Current Protocols inMolecular Biology, 1994—current, John Wiley & Sons. The term“recombinant” when used with reference, e.g., to a cell, or nucleicacid, protein, or vector, indicates that the cell, organism, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells may express genes thatare not found within the native (non-recombinant or wild-type) form ofthe cell or express native genes that are otherwise abnormallyexpressed-over-expressed, under-expressed or not expressed at all.

The terms “transgenic”, “transformed”, “transformation”, “transformed”and “transfection” are similar in meaning to “recombinant”.“Transformation”, “transgenic”, and “transfection” refer to the transferof a polynucleotide into the genome of a host organism or into a cell.Such a transfer of polynucleotides can result in genetically stableinheritance of the polynucleotides or in the polynucleotides remainingextra-chromosomally (not integrated into the chromosome of the cell).Genetically stable inheritance may potentially require the transgenicorganism or cell to be subject for a period of time to one or moreconditions which require the transcription of some or all of transferredpolynucleotide in order for the transgenic organism or cell to liveand/or grow. Polynucleotides that are transformed into a cell but arenot integrated into the host's chromosome remain as an expression vectorwithin the cell. One may need to grow the cell under certain conditionsin order for the expression vector to remain in the cell or the cell'sprogeny. Further, for expression to occur the organism or cell may needto be kept under certain conditions. Host organisms or cells containingthe recombinant polynucleotide can be referred to as “transgenic” or“transformed” organisms or cells or simply as “transformants”, as wellas recombinant organisms or cells.

A method of selecting an isolated polynucleotide that affects the levelof expression of a polypeptide in a virus or in a host cell (eukaryotic,such as plant, yeast, fungi, or algae; prokaryotic, such as bacteria)may include the steps of: constructing an isolated polynucleotide of thepresent invention; introducing the isolated polynucleotide into a hostcell: measuring the level of a polypeptide in the host cell containingthe isolated polynucleotide; and comparing the level of a polypeptide inthe host cell containing the isolated polynucleotide with the level of apolypeptide in a host cell that does not contain the isolatedpolynucleotide.

An “expression cassette” is a nucleic acid construct, typicallygenerated recombinantly or synthetically, which comprises a series ofspecified nucleic acid elements that permit transcription of aparticular nucleic acid in a host cell. Typically, an expressioncassette is part of an “expression vector”. An expression vector orsimply a “vector” is nucleic acid capable of replicating in a selectedhost cell or organism. An expression vector can replicate as anautonomous structure, or alternatively can integrate into the host cellchromosomes or the nucleic acids of an organelle, and thus replicatealong with the host cell genome. Thus, an expression vector arepolynucleotides capable of replicating in a selected host cell,organelle, or organism, e.g., a plasmid, virus, artificial chromosome,nucleic acid fragment, and for which certain genes on the expressionvector are transcribed and translated into a polypeptide or proteinwithin the cell, organelle or organism: or any suitable construct knownin the art, which comprises an “expression cassette”.

The term “capable of hybridizing under stringent hybridizationconditions” refers to annealing a first nucleic acid to a second nucleicacid under stringent hybridization conditions (defined below). In anexemplary embodiment, the first nucleic acid is a test sample, and thesecond nucleic acid is the sense or antisense strand of a nucleic acidof interest. Hybridization of the first and second nucleic acids isconducted under standard stringent conditions, e.g., high temperatureand/or low salt content, which tend to disfavor hybridization ofdissimilar nucleotide sequences.

Any expression vector containing the polynucleotides described hereinoperably linked to a promoter is also covered by this invention. Apolynucleotide sequence is operably linked to an expression controlsequence(s) (e.g., a promoter and, optionally, an enhancer) when theexpression control sequence controls and regulates the transcription andtranslation of that polynucleotide sequence. An expression vector is areplicon, such as plasmid, phage or cosmid, and which contains thedesired polynucleotide sequence operably linked to the expressioncontrol sequence(s). The promoter may be, or is identical to, a viralphage, bacterial, yeast or other fungal, insect, plant, or mammalianpromoter. Similarly, the enhancer may be the sequences of an enhancerfrom virus, phage, bacteria, blue-green algae, yeast or other fungi,insects, plants, or mammals.

“Operably linked” refers to the association of two or more nucleic acidfragments on a single polynucleotide so that the function of one isaffected by the other. For example, a promoter is operably linked with acoding sequence so that the promoter is capable of affecting theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in sense or antisenseorientation. When a promoter is operably linked to a polynucleotidesequence encoding a protein or polypeptide, the polynucleotide sequenceshould have an appropriate start signal (e.g., ATG) in front of thepolynucleotide sequence to be expressed. Further, the sequences shouldbe in the correct reading frame to permit transcription of thepolynucleotide sequence under the control of the expression controlsequence and, translation of the desired polypeptide or protein encodedby the polynucleotide sequence. If a gene or polynucleotide sequencethat one desires to insert into an expression vector does not contain anappropriate start signal, such a start signal can be inserted in frontof the gene or polynucleotide sequence. In addition, a promoter can beoperably linked to a RNA gene encoding a functional RNA.

A wide variety of promoters are known to those of ordinary skill in theart as are other regulatory elements that can be used alone or incombination with promoters. A wide variety of promoters that directtranscription in plants cells can be used in connection with the presentinvention. For purposes of describing the present invention, promotersare divided into two types, namely, constitutive promoters andnon-constitutive promoters. Constitutive promoters are classified asproviding for a range of constitutive expression. Thus, some are weakconstitutive promoters, and others are strong constitutive promoters.Non-constitutive promoters include tissue-preferred promoters,tissue-specific promoters, cell-type specific promoters, andinducible-promoters.

Of interest in certain embodiments of the present invention areinducible-promoters for plants that respond to various forms ofenvironmental stresses, or other stimuli, including, for example,mechanical shock, heat, cold, salt, flooding, drought, salt, anoxia,pathogens, such as bacteria, fungi, and viruses, and nutritionaldeprivation, including deprivation during times of flowering and/orfruiting, and other forms of plant stress. For example, the promoter canbe induced by one or more, but not limiting to one of the following:abiotic stresses such as wounding, cold, dessication, ultraviolet-B (vanDer Krol, et al., Plant Physiol. 121:1153-1162 (1999)), heat shock(Shinmyo, et al., Biotechnol. Bioeng. 58:329-332 (1998)) or other heatstress, drought stress, or water stress. The promoter may further be oneinduced by biotic stresses, including pathogen stress, such as stressinduced by a virus (Sohal et al., Plant Mol. Biol. 41:75-87 (1999)) orfungi (Eulgem, et al., Embo J. 18:4689-4699 (1999); Cormack, et al.,Biochim Biophys Acta 1576:92-100 (2002)); stresses induced as part ofthe plant defense pathway (Lebel, et al., Plant J. 16:223-33 (1998)); orpromoters induced by other environmental signals, such as light (Ngai,et al., Plant J. 12:1021-1034 (1997)), carbon dioxide (Kucho, et al.,Plant Physiol. 121:1329-1338 (1999); Kucho, et al., Plant Physiol.133:783-7893 (2003)), hormones or other signaling molecules such asauxin, hydrogen peroxide and salicylic acid (Chen, et al., Plant J.19:667-677 (1999); Chen, et al., Plant J. 10:955-966 (1996)), sugars andgibberellin (Lu, et al., J. Biol. Chem. 273:10120-10131 (1998)) orabscissic acid and ethylene (Leubner-Metzger, et al., Plant Mol. Biol.38:785-795 (1998)).

In other embodiments of the invention, tissue-specific promoters areused. Tissue-specific expression patterns as controlled by tissue- orstage-specific promoters that include, but are not limited to,fiber-specific, green tissue-specific, root-specific, stem-specific,root-specific, and flower-specific. Examples of the utilization oftissue-specific expression includes, but is not limit to, the expressionin leaves of the desired peptide for the protection of plants againstfoliar pathogens, the expression in roots of the desired peptide for theprotection of plants against root pathogens, and the expression in rootsor seedlings of the desired peptide for the protection of seedlingsagainst soil-borne pathogens. In many cases, however, protection againstmore than one type of pathogen may be sought, and expression in multipletissues will be desirable. Another example of promoters that areexpressed in specific tissue are chlorophyll A/B binding protein (CAB)promoter (Bansal, et al., Proc. Natl. Acad. Sci. USA 89(8):3654-8(1992)), small subunit of ribulose-1,5-bisphosphate carboxylase (ssRBCS)promoter (Bansal, et al., Proc. Natl. Acad. Sci. USA 89(8):3654-8(1992)), phosphoenolpyruvate carboxylase 1 (PPC1) promoter (Kausch, etal., Plant Mol. Biol. 45(1):1-15 (2001)), a senescence activatedpromoter, SEE1, (Robson, et al., Plant Biotechnol. J. 2(2): 101-12(2004)), and the sorghum leaf primoridia specific promoter, RS2,(GenBank Accession No. E1979305.1).

Although many promoters from dicotyledons have been shown to beoperational in monocotyledons and vice versa, in a majority of casesdicotyledonous promoters are selected for expression in dicotyledons,and monocotyledonous promoters are selected for expression inmonocotyledons. There are also promoters that control expression ofgenes in green tissue or for genes involved in photosynthesis from bothmonocotyledons and dicotyledons such as the maize from the phosphoenolpyruvate carboxylase gene (Hudspeth, et al., Plant Physiol. 98:458-464(1992)). There are suitable promoters for root specific expression (deFramond, FEBS Lett. 290:103-106 (1991): Hudspeth, et al., Plant Mol.Biol. 31:701-705 (1996)).

A promoter selected can be an endogenous promoter, i.e., a promoternative to the species and or cell type being transformed. Alternatively,the promoter can be a foreign promoter, which promotes transcription ofa length of DNA of viral, microbes, bacterial or eukaryotic origin,invertebrates, vertebrates including those from plants and plantviruses. For example, in certain embodiments, the promoter may be ofviral origin, including a cauliflower mosaic virus promoter (CaMV), suchas CaMV 35S or 19S, a figwort mosaic virus promoter (FMV 35S), or thecoat protein promoter of tobacco mosaic virus (TMV). The promoter mayfurther be, for example, a promoter for the small subunit ofribulose-1,3-bisphosphate carboxylase. Promoters of bacterial origininclude the octopine synthase promoter, the nopaline synthase promoterand other promoters derived from native Ti plasmids could also be(Herrera-Estrella, et al., Nature 303:209-213 (1983)). Some of thesepromoters are constitutive promoters for plants.

The promoters may be such that they are activated by other elementsknown to those of ordinary skill in the art, so that production of theprotein encoded by the recombinant nucleic acid sequence may beregulated as desired. In one embodiment of the invention, a DNAconstruct containing a non-constitutive promoter operably linked to apolynucleotide encoding the desired polypeptide of the invention is usedto make a transformed plant that selectively increases the level of thedesired polypeptide of the invention in response to a signal. The term“signal” refers to a condition, stress or stimulus that results in orcauses a non-constitutive promoter to direct expression of the codingsequence operably linked to it. To make such a transformed plant inaccordance with the invention, a DNA construct is provided that includesa non-constitutive promoter operably linked to a polynucleotide encodingthe desired polypeptide of the invention. The construct is incorporatedinto a plant genome to provide a transformed plant that expresses thepolynucleotide in response to a signal.

In another embodiment of the invention, a DNA construct comprising aSbPRH1 or SbPRH2 or both SbPRH1 and SbPRH2 operably linked to promotersthat are active in root hair cells are used to make a transformed plantthat selectively increases the transcript or mRNA of the desiredenzyme(s) of the invention in root hair cells. However, other promotersmay be used in this invention. It is understood to those of ordinaryskill in the art that the regulatory sequences that comprise a plantpromoter driven by RNA polymerase II reside in the region approximately2900 to 1200 base pairs up-stream (5′) of the translation initiationsite or start codon (ATG). For example, the full-length promoter for thenodule-enhanced PEP carboxylase from alfalfa is 1277 base pairs prior tothe start codon (Pathirana, et al., Plant J. 12:293-304 (1997)), thefull-length promoter for cytokinin oxidase from orchid is 2189 basepairs prior to the start codon (Yang, et al., J. Exper. Bot.53:1899-1907 (2002)), the full-length promoter for ACC oxidase frompeach is 2919 base pairs prior to the start codon (Moon, et al., J.Exper. Bot. 55:1519-1528 (2004)), full-length promoter for cytokininoxidase from orchid is 2189 base pairs prior to the start codon,full-length promoter for glutathione peroxidase1 from Citrus sinensis is1600 base pairs prior to the start codon (Avsian-Kretchmer, et al.,Plant Physiol. 135:1685-1696 (2004)), and the full-length promoter forglucuronosyltransferase from cotton is 1647 base pairs prior to thestart codon (Wu, et al., Cell Research 17:174-183 (2007)). Mostfull-length promoters are 1700 base pairs prior to the start codon. Theaccepted convention is to describe this region (promoter) as −1700 to−1, where the numbers designate the number of base pairs prior to the“A” in the start codon.

One aspect of this invention is the transformation of fungi with thegenes described here so that the transformed fungi can produce the geneproducts and make the compounds described herein. While one can use thesome or all of the various expression vectors described herein togenerate transformed fungi, one may need to use promoters that work infungi. While some of the plant promoters described above may induceexpression of foreign genes in fungi, one may want to use promoters thatexist in fungi. It should not be surprising to one of ordinary skill inthe art that fungal promoters, similar to plant and bacteria promoters,can be constitutive or inducible. Non-limiting examples of constitutivepromoters include the following: (1) Pna2/TPI which is a hybrid promotercontaining sequences from the Aspergillus niger neutral amylase promoterand the A. nidulans triose phosphate isomerase promoter (Olempska-Beer,et al., Regul. Toxicol. Pharm 45: 144-158 (2006)); (2) GpdA (aka GAPDH),the A. nidulans glyceraldehyde 3-phosphate dehydrogenase promoter (Punt,et al., J. Biotechnol. 17:19-34 (1991)); (3) TrpC, an A. nidulanstryptophan biosynthesis promoter (Hamer and Timberlake, Mol. Cell Biol.7(7):2352-9 (1987)); and ToxA and ToxB promoters obtained fromPyrenophora tritici-repentis capable of driving expression of genes onplasmids in a variety of filamentous fungi (Andrie, et al., Mycologia97:1152-1161 (2005). Additional constitutive promoters for Saccharomycescerevisiae include, but are not limited to, Cyc, Adh, Ste5, Pgk, Gpd,Clb, Aox1, His4, and Tef promoters.

Non-limiting examples of inducible fungal promoters include thefollowing: (1) TAKA-A amylase promoter from A. oryzae (Tada, et al.,Mol. Gen. Genet. 229:301-306 (1991): (2) the α-amylase B (AmyB) promoterfrom A. orvzae (Hoshida, et al., Biosci. Biotechnol. Biochem.69(6):1090-7 (2005): (3) the glucoamylase A (GlaA) promoter from A.niger (Ganzlin and Rinas. J. Biotechnol. 135:266-271 (2008); (4) thealcohol regulon promoters in A. nidulans (AlcA-alcohol dehydrogenase;AldA—aldehyde dehydrogenase; and positive regulator AlcR) (Gwynne, etal., Biochem. Soc. Trans. 17:338-340 (1989); and U.S. Pat. No.5,624,046); (5) the A. awamori endoxylanase promoter (Ex/A) (Gouka, etal., Appl. Microbiol. Biotechnol. 46:28-35 (1996); (6) the A. fumigaltusnitrite reductase promoter (NiiA) (Amaar and Moore, Curr. Genet.33:206-215 (1998)); (7) the Trichoderma reesei cellobiohydrolase Ipromoter (Cbhl) (Harkki, et al., Enzyme Microb. Technol. 13:227-233(1991); the Schizosaccharomyces pombe high affinity copper transporter(Ctr4) (Bellemare, et al., Gene 273:191-198 (2001)); and the A. orvzaethiamine biosynthesis promoter (ThiA) (Shoji, et al., FEMS Microbiol.Lett. 244:41-46 (2005)). Additional inducible promoters for S.cerevisiae include Met25, Gal1, LacZ, and Kladh4. Other examples ofinducible promoters are those for the heat shock, alcohol dehydrogenase,and glucocorticoid response element genes that are activated by heat,alcohol and steroid hormones respectively.

Examples of other fungal promoters include the budding yeast enolasegene (Eno1) promoter (U.S. Pat. No. 7,999,090), the Pho5 promoter (U.S.Pat. No. 7,811,823), UAS_(MAL) which is a bidirectional promoter elementrequired for the expression of both the MAL61 and MAL62 genes of theSaccharomyces MAL6 locus (Levine, et al., Current Genetics 181-189(1992), the bidirectional maltase gene promoter in Kluyveromyces lactis(U.S. Pat. No. 6,596,513), the bidirectional PcbAB-PcbC promoterAcremonium chrysogenum (Menne, et al., Appl. Microbiol. Biotechnol.42:57-66 (1994)), the 3-phosphoglycerate kinase promoter (U.S. Pat. No.5,646,012), the RP28 ribosomal protein promoter (U.S. Pat. No.5,627,049), the transaldolase promoter (U.S. Pat. No. 5,616,474), thepyruvate decarboxylase promoter (U.S. Pat. No. 5,631,143), and thebidirectional promoter of α-aminoadipyl-cysteinyl-valine [ACV]synthetase/isopenicillin N-synthetase from Acremonium chrysogenum (Meme,et al., Appl. Microbiol. Biotechnol. 42(1):57-66 (1994)).

This invention can include recombinant bacterium is Escherichia coli,Bacillus subtilus, or Pseudomonas spp. that express the genes ofinterest and produce the encoded proteins (enzymes). Non-limitingexamples of suitable bacterial promoters include promoters capable ofrecognizing the T4, T3, Sp6 and T7 polymerases, the P_(R) and P_(L)promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5,tac, lpp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.subtilis, the promoters of the bacteriophages of Bacillus spp., the intpromoter of bacteriophage lambda, the bla promoter of pBR322, and theCAT promoter of the chloramphenicol acetyl transferase gene. Prokaryoticpromoters have been reviewed by Glick, Ind. Microbiol. 1:277 (1987),Watson, et al., Molecular Biology of the Gene, 4^(th) ed. (BenjaminCummins 1987), and by Ausubel, et al. (1994).

The present invention also includes transforming blue-green algae withthe DNA and expression vectors, expressing the genes, and producing thegene products and compounds described herein. As such, suitablepromoters for blue-green algae include, but are not limited to, thepromoters for rrnA and rrnB operons (Kumano, et al., Molecular andGeneral Genetics MGG 202(2): 173-178 (1986)), the promoter for RuBisCO(ribulose-1,5-diphosphate carboxylase/oxygenase) gene (U.S. Pat. No.5,804,408), the RNA polymerase promoter from cyanophage Syn5 (Zhu, etal., J. Biol. Chem. 288(5):3545-3552 (2013)), the promoter of the AbrB2gene (Dutheil, et al., J. Bacteriol. 194(19):5423-5433 (2012)), thepsbA2 promoter from Synechocystis PCC6803 (Lindberg, et al., MetabolicEngineering 12:70-79 (2009)), and high light-inducible promoter for highlight-inducible polypeptides (hliA, hliB and hliC) from SynechocystisPCC6803 (He, et al., J. Biol. Chem. 276(1): 306-314 (2001)).

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A reference sequence isa defined sequence used as a basis for a sequence comparison; areference sequence may be a subset of a larger sequence, or genesequence given in a sequence listing.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., 85% identity, 90% identity, 99%, or 100% identity), when comparedand aligned for maximum correspondence over a comparison window, ordesignated region as measured using a sequence comparison algorithm orby manual alignment and visual inspection.

The phrase “substantially identical”, in the context of twopolynucleotides or polypeptides, refers to two or more sequences orsubsequences that have at least about 85%, identity, at least about 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. In an exemplary embodiment, asubstantial identity exists over a region of the sequences that is atleast about 50 residues in length. In another exemplary embodiment, asubstantial identity exists over a region of the sequences that is atleast about 100 residues in length. In still another exemplaryembodiment, a substantial identity exists over a region of the sequencesthat is at least about 150 residues or more in length. In one exemplaryembodiment, the sequences are substantially identical over the entirelength of the nucleic acid or protein sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from about 20 to about 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch. J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT. FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Ausubel et al., eds., Current Protocols in Molecular Biology, 1995supplement).

An exemplary algorithm for sequence comparison is PILEUP. PILEUP createsa multiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package. e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984)).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., 1990). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively hybridizes to” or “specifically hybridizes to”refers to the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA). In general two nucleic acid sequences are saidto be “substantially identical” when the two molecules or theircomplements selectively or specifically hybridize to each other understringent conditions.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Hybridization with Nucleic Probes Parts I andII, Elsevier (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For high stringencyhybridization, a positive signal is at least two times background,preferably 10 times background hybridization. Exemplary high stringencyor stringent hybridization conditions include: 50% formamide, 5×SSC and1% SDS incubated at 42° C. or 5×SSC and 1% SDS incubated at 650° C.,with a wash in 0.2×SSC and 0.1% SDS at 65° C. However, other highstringency hybridization conditions known in the art can be used.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This situation can occur, forexample, when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code. In such cases, thenucleic acids typically hybridize under moderately stringenthybridization conditions. Exemplary “moderately stringent hybridizationconditions” include hybridization in a buffer of 40% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positivehybridization is at least twice background. Those of ordinary skill willreadily recognize that alternative hybridization and wash conditions canbe utilized to provide conditions of similar stringency.

Oligonucleotides and polynucleotides that are not commercially availablecan be chemically synthesized e.g., according to the solid phasephosphoramidite triester method first described by Beaucage andCaruthers. Tetrahedron Letts. 22:1859-1862 (1981), or using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Other methods for synthesizing oligonucleotides andpolynucleotides are known in the art. Purification of oligonucleotidesis by either native acrylamide gel electrophoresis or by anion-exchangeHPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981). Using of machines for sequencing DNA or RNA is known in the artfield.

This invention utilizes routine techniques in the field of molecularbiology. Basic texts disclosing the general methods of use in thisinvention include Green and Sambrook, 4th ed. 2012, Cold Spring HarborLaboratory; Kriegler, Gene Transfer and Expression: A Laboratory Manual(1993); and Ausubel et al., eds., Current Protocols in MolecularBiology, 1994—current, John Wiley & Sons. Unless otherwise noted,technical terms are used according to conventional usage. Definitions ofcommon terms in molecular biology maybe found in e.g., Benjamin Lewin,Genes IX, published by Oxford University Press, 2007 (ISBN 0763740632):Krebs, et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The methods described above may be applied to transform a wide varietyof plants, including decorative or recreational plants or crops, but areparticularly useful for treating commercial and ornamental crops.Examples of plants that may be transformed in the present inventioninclude, but are not limited to, Acacia, alfalfa, aneth, apple, apricot,artichoke, arugula, asparagus, avocado, banana, barley, beans, beech,beet, Bermuda grass, blackberry, blueberry, blue grass, broccoli,brussels sprouts, cabbage, camelina, canola, cantaloupe, carrot,cassava, cauliflower, celery, cherry, chicory, cilantro, citrus,clementines, coffee, corn, cotton, cucumber, duckweed, Douglas fir,eggplant, endive, escarole, eucalyptus, fennel, fescue, figs, foresttrees, garlic, gourd, grape, grapefruit, honey dew, jatropha, jicama,kiwifruit, lettuce, leeks, lemon, lime, loblolly pine, maize, mango,melon, mushroom, nectarine, nut, oat, okra, onion, orange, an ornamentalplant, palm, papaya, parsley, pea, peach, peanut, pear, pepper,persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato,pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry,rice, rye, rye grass, scallion, sorghum, southern pine, soybean,spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweetpotato, sweetgum, switchgrass, tangerine, tea, tobacco, tomato, turf,turnip, a vine, watermelon, wheat, yams, and zucchini. Other suitablehosts include blue-green algae and other photosynthetic organisms, evenif these organisms are not considered plants.

The term “plant” includes whole plants, plant organs, and progeny ofsame. Plant organs comprise, e.g., shoot vegetative organs/structures(e.g., leaves, stems and tubers), roots, flowers and floralorgans/structures (e.g., bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat)and fruit (the mature ovary), plant tissue (e.g., vascular tissue,ground tissue, and the like) and cells (e.g., guard cells, egg cells,trichomes and the like). The class of plants that can be used in themethod of the invention is generally as broad as the class of higher andlower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns, and multicellular algae. It includes plants of a variety ofploidy levels, including aneuploid, polyploid, diploid, haploid andhemizygous.

Some exemplary blue-green algae that can be transformed with the vectorsand DNA described herein and that can produce the gene products andcompounds discussed herein include, but are not limited to, Anacvystisnidulans, Synechococcus spp., Svnechocystis spp., Spirulina platensis,Anabaena PCC7120, Nostoc PCC7119, and Calothrix PCC7601. Transformationof blue-green algae with the expression vectors and DNA described hereincan be carried out using methods known to one of ordinary skill in theart, such as those methods described in Porter, CRC Critical Reviews inMicrobiology 13(2):111-132 (1986); Lightfoot, et al., J. GeneralMicrobiology 134:1509-1514 (1988); Daniell, et al., Proc. Natl. Acad.Sci. USA 83:2546-2550 (1986). Matsunaga, et al., Appl. Biochem.Biotechnol. 24/25:151-160 (1990) and Liu, et al., Proc. Natl. Acad. Sci.USA 108(17):6905-6908 (2011)))). It is recognized that blue-green algaeare cyanobacteria and thus are considered bacteria. As discussed suprathis invention includes bacteria transformed with the polynucleotides ofthis invention and using the recombinant bacteria to produce the encodedproteins and make the compounds disclosed herein.

In addition to plants, the polynucleotides of this invention can betransformed into fungi and the encoded proteins (enzymes) can beproduced by the fungi. Non-limiting examples of suitable fungiSaccharomvces spp., Pichia spp., Candida spp., Aspergillus spp., orKluvveromyces spp. More specifically, one can utilize S. cerevisiae. P.pastoris, P. methanolica. C. albicans, A. niger, or Kluvveromyces lactisfor this invention.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples and theaccompanying drawings, which are included herein only to furtherillustrate the invention and are not intended to limit the scope of theinvention as defined by the claims. The examples and drawings describeat least one, but not all embodiments, of the inventions claimed.Indeed, these inventions may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements.

Example 1 Identification of ESTs with Putative Cytochrome P450 Domains

Sorghum root-hair EST database mining is performed as described inBaerson, et al., J. Biol. Chem. 283:3231-3247 (2008) and Cook, et al.,(2010). All ESTs have been deposited in GenBank and have beenincorporated into the current NCBI unigene release (build #27, 2 Mar.2008). Fourteen cytochrome P450-like sequences on ESTs are identified byBLASTN and TBLASTN analysis (Altschul, et al., (1997)). These sequencesare assembled into eleven unique sequences by cluster analysis, eight ofwhich are represented by a single EST (i.e., singletons).

Example 2 Identification of Root Hair Cell Expressed Putative CytochromeP450 Genes

To identify the cytochrome P450 sequences where are expressedspecifically or predominantly in root hair cells of S. bicolor,quantitative real-time PCR (qRT-PCR) is performed using cDNAs preparedfrom total RNA isolated from root hairs, root systems, developingpanicles, stems, immature and fully expanded leaves, and shoot apices.S. bicolor seeds are purchased from Crosbyton Seed Company (Crosbyton,Tex.) and grown according to conditions set forth in Cook, et al.(2010). Mature leaves, stems and emerging panicles are harvested fromapproximately two-month old, greenhouse-grown S. bicolor plants.Immature leaves and shoot apices are isolated from eight-day oldseedlings maintained in a growth chamber at 28° C., 16 hours light and 8hours dark. 400 μmol/m² sec intensity. Total root systems and root hairsare isolated from 8-day old seedlings grown using a capillary matsystem. All tissue collected is flash-frozen in liquid nitrogen and keptat −80° C. until RNA extraction. Root hairs are isolated according tothe protocol set forth in Bucher, et al., Plant Mol. Biol. 35:497-508(1997). Total RNA for qRT-PCR and cDNA cloning are extracted from thepreviously flash-frozen S. bicolor tissues using TRIzol® Reagent (LifeTechnologies. Carlsbad, Calif.) using the protocol described in Cook, etal., 2010. Briefly, frozen plant tissues are homogenized using ahandheld homogenizer (homogenization step of 30 seconds at 25.000 rpm)which is followed by RNA purification with an RNeasy Plant Mini Kit(Qiagen, Valencia, Calif.) according to the manufacturer's instructions.Isolated RNAs are further treated with RNase-free DNase to removeresidual DNA contamination (Qiagen, Valencia, Calif.). The purity ofRNAs is determined spectrophotometrically, and the integrity of purifiedRNAs is also assessed by agarose gel electrophoresis, qRT-PCR reactionsare performed in triplicate using a model 7300 Sequence Detection System(Applied Biosystems, Carlsbad, Calif.) as previously described (Cook, etal., 2010). PCR primers were designed using Primer Express® v2.0software (Applied Biosystems, Foster City, Calif.) and the Amplifyprogram (Engels, W. R. 1993. Trends Biochem. Sci. 18: 448-450). Adissociation curve is generated at the end of each PCR cycle to verifythat a single product is amplified using software provided with model7300 Sequence Detection System. A negative control reaction minus cDNAtemplate (non-template control) is also routinely performed intriplicate for each primer pair. The change in fluorescence of SYBR®Green I dye in every cycle is monitored by the GenAmp® 7300 systemsoftware, and the threshold cycle (C_(T)) above background for eachreaction is calculated. The C_(T) value of 18S rRNA is subtracted fromthat of the gene of interest to obtain a ΔC_(T) value. The C_(T) valueof an arbitrary calibrator (e.g., the tissue sample from which thelargest ΔC_(T) values are obtained) is subtracted from the ΔC_(T) valueto obtain a ΔΔC_(T) value. The fold-changes in expression level relativeto the calibrator are calculated as 2^(−ΔΔCT). The gene specific PCRprimer pairs used for the 18s rRNA and three candidate P450s are listedin Table 1 below.

TABLE 1 Primer pairs  Primer Name (5′ -> 3′) 21G12 RTHAIR1_21_G12.g1_AAGATCCAAGGCTACCATG forward A002 TGC (SEQ ID NO: 1) 21G12RTHAIR1_21_G12.g1_ AACGTTGGCGACGACTTAT reverse A002 TG (SEQ ID NO: 2)69C05 RTHAIR1_69_C05.g1_ CCACTTTGATTGGTCCCTG forward A002C (SEQ ID NO: 3) 69C05 RTHAIR1_69_C05.g1_ TCTGTCATGTCAACCTCAT reverseA002 CGAC (SEQ ID NO: 4) 18S 18s rRNA GGCTCGAAGACGATCAGAT forwardACC (SEQ ID NO: 5) 18S 18s rRNA TCGGCATCGTTTATGGTT  reverse(SEQ ID NO: 6)

The sequences covered by each of these three primer sets arepreferentially expressed in root hair cells and slightly less so in rootsystems. See FIG. 2 for levels of expression in various plant tissue asdetermined by qRT-PCR.

Example 3 Generation of cDNA of the Root Hair Cell Expressed Genes

To obtain full-length cDNA clones of the three root hair cell expressedgenes, partial sequences for S. bicolor RTHAIR1_21_G12.gl_A002 andRTHAIR1_69_C05.gl_A002 (designated 21G12 and 69C05, respectively)obtained from previously generated root hair EST assemblies (Baerson, etal. (2008)) are used for both 5′- and 3′ rapid amplification of cDNAends (RACE) using the BD SMART™ RACE cDNA Amplification Kit (Clontech,Palo Alto, Calif.) according to the manufacturer's instructions. Primersets for 5′- and 3′-RACE are as follows: for 21G12, toward the 5′ endprimer 5′-TGGGACGAACGGGGCAGGAG-3′ (SEQ ID NO: 7) and toward the 3′ endprimer 5′-GGCATCAAGATCCAAGGCTAC-3′ (SEQ ID NO: 8); and for 69C05, towardthe 5′ end primer 5′-TCATGTCAACCTCATCGACACC-3′ (SEQ ID NO: 9) and towardthe 3′ end primer 5′-TCTACCACITGATTGGTCCCTG-3′ (SEQ ID NO: 10).Full-length cDNAs are then amplified with primer pairs complementary tothe 5′- and 3′-UTRs identified in RACE experiments using PfuUltra DNApolymerase (Stratagene, La Jolla, Calif.) and first-strand cDNAgenerated from RNA extracted from sorghum root hairs. Severalindependent isolates from each amplification are sequenced to ensure theauthenticity of the open reading frames. The DNA sequence of SbPRH1 isin SEQ ID NO: 11, and the deduced amino acid sequence is in SEQ ID NO:12. The DNA sequence of SbPRH2 is in SEQ ID NO: 13, and the deducedamino acid sequence is in SEQ ID NO: 14.

The amino acid sequences which are deduced from SbPRH1 and SbPRH2 are40.0% identical to each other. BLASTP analysis of these two amino acidsequences reveal that neither sequences exhibit extensive similarity toknown functionally characterized plant P450 sequences. The amino acidsequences of SbPRH1 and SbPRH2 correspond to typical plant P450 familymembers of the CYP71 class, i.e., CYP71AM1 and CYP71V7, respectively.These nomenclatures will be used for these genes hereafter. Thepredicted amino acid sequences of the cDNA clones exhibit all of themajor elements typically found in plant cytochrome P450 monooxygenases,such as the proline-rich region, the O₂ binding site, and the PERF-motiflocated upstream of the heme-binding cysteine motif (Bak, et al.,“Cytochrome P450” in The Arabidopsis Book v.9 ISSN: 1543-8120 publishedby The American Society of Plant Biologists (2011)).

A third party previously performed genomic sequencing of S. bicolor anddeposited the sequence of putative genes and putative proteins inGenBank. The DNA sequences of SbPRH1 (CYP71AM1) is similar to the DNAsequence of a hypothetical gene at GenBank accession numberXM_002451987, and the putative amino acid sequences of the two arecompletely identical. The DNA sequences of SbPRH2 (CYP71V7) is similarto the DNA sequence of a hypothetical gene at GenBank accession numberXM_002465028, and the putative amino acid sequences of the two arecompletely identical. It is worth noting that the investigators whodeposited the sequences in GenBank did not perform experiments toconfirm that the presumed DNA sequence actually is a full-length geneand is transcriptionally active in S. bicolor. Nor did the investigatorsdetermine the activity of the putative protein. In light of theinformation contained in Example 7 below, simply identifying a putativegene encoding a putative cytochrome P450 does not mean that one knowsthe activity of the encoded enzyme.

Example 4 Recombinant Constructs for Heterologous Expression in S.cerevisiae

To examine whether these sorghum P450s possesses dihydroxylationactivity towards 3-methyl-5-pentadecatrienyl resorcinol, a methylatedresorcinolic intermediate in the sorgoleone biosynthetic pathway, oneneeds to perform functional expression in yeast. Two plasmids, pYeG12carrying CYP71AM1 and pYeC05 carrying CYP71V7, are constructed byinserting the full-length cDNA into the yeast expression vector pYeDP60plasmid that allows galactose-inducible expression of P450s in yeast(Pompon, et al. (1996)). For heterologous expression in yeast, openreading frames are amplified using PfuUltra DNA polymerase (Stratagene,La Jolla, Calif.) and cloned in the pYeDP60 vector (Pompon, et al.,Methods Enzymol. 272:51-64 (1996)) using the BamHI and KpnI restrictionsites, yielding the plasmids pYeG12 (for 21G12) and pYeC05 (for 69C05).For pYeG12, the forward primer is CYP71AM1F35′-TACCATGGACGAATACTTTGTTGACCTGC-3′ (SEQ ID NO: 15), and the reverseprimer is CYP71AM1R3 5′-TIATGCATCAATCGATGCAGCAGCTG-3′ (SEQ ID NO: 16).For pYeC05, the forward primer is CYP71V7F35′-TACCATGGAAGTGTTCCAACCCCTCC-3′ (SEQ ID NO: 17), and the reverse primeris CYP71V7R3 5′-CTAGCTTGGCACCCTGCTGGTTTC-3′ (SEQ ID NO: 18). Theconstructs are then transformed into the S. cerevisae WAT11 strain(Pompon, et al. (1996)) using the lithium acetate method (see Burke, etal., Methods in Yeast Genetics, (Cold Spring Harbor Labs, N Y, 2000).Briefly, yeast are grown overnight in a 30° C. shaker in 10 ml completeYPGA liquid medium [10 g/L yeast extract (Difco), 10 g/L bactopeptone(Difco) 20 g/L glucose, 200 mg/L adenine] to an OD₆₀₀ between 1.0 and1.5. The cells are collected by centrifugation at 2500×g for 5 minutesat 4° C., and are resuspended in 1.5 ml of a 0.1 M lithium acetate(LiAc) solution in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). A 0.12ml aliquot of cells in a 1.5-ml Eppendorf tube is used fortransformation. Plasmid DNA (1 μg in 10 μl TE buffer) and salmon spermDNA as DNA carrier (100 μg from a 10 mg/ml solution in TE after 5minutes boiling) are added to the competent yeast cells. 500 μl of 40%polyethylene glycol 3350 in 0.1 M LiAc solution in TE buffer and 57 μlDMSO are added. The mixture is incubated for 15 minutes at roomtemperature, then for 15 minutes at 42° C. After centrifugation for 10seconds at 10,000×g the transformed yeast cells are resuspended in 200μl TE buffer, and then are plated on SGI minimal media [7 g/L yeastnitrogen base; 20 g/L glucose; 20 mg/L tryptophan; 20 g/L agar (Difco)].All yeast transformants are confirmed by colony-PCR using gene-specificprimers, and by further restriction analyses performed using isolatedplasmid preparations.

Example 5 Preparation of Yeast Microsomes

Microsomes are prepared according to the method described by Pompon, etal. (1996). Colonies from strains transformed with pYeDP60 and the twoP450 constructs are transferred using sterile toothpicks into 15 ml SGIminimal media (1 g/L bacto casamino acids, 7 g/L yeast nitrogen base, 20g/L glucose, and 20 mg/L tryptophan) and are grown overnight at 30° C.to a density of 6×10⁷ cells per ml. This pre-culture is diluted into 250ml YPGE (5 g/L glucose, 10 g/L yeast extract, 10 g/L bactopeptone, 3%[v/v]ethanol) to a density of 2×10⁵ cells per ml, and is grown until itreaches a density of 8×10⁷ cells per ml (approximately 30 h). Theculture is centrifuged, and the pellet is resuspended in 250 ml YPIinduction medium (10 g/L yeast extract, 10 g/L bactopeptone 20 g/Lgalactose) and grown for 16 hours at 30° C. Microsomal membranes areisolated after mechanical disruption of yeast cells with glass beads(Pompon, et al. (1996)). Microsomal protein concentrations aredetermined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, Calif.)with bovine serum albumin as a standard. Dithionite-reduced, carbonmonoxide difference spectra are obtained using an Evolution 300spectrophotometer (Thermo Scientific, Somerset, N.J.) according to themethod described by Guengerich, et al., Nat. Protoc. 4:1245-1251 (2009).Microsomes from yeast transformed with vector pYeDP60 alone are used asnegative controls.

The recombinant proteins are expressed in the S. cerevisae WAT11 strainthat co-expresses the NADPH-cytochrome P450 reductase gene fromArabidopsis thaliana (Urban, et al., Biochimie 72:463-472 (1990);Pompon, et al. (1996)). The P450 functional expression is determined at450 nm using a carbon monoxide (CO) differential spectrum. The COdifference spectrum with a characteristic peak at 450 nm is obtainedfrom microsomal fractions extracted from yeast expressing CYP71AM1 andCYP71V7, indicating the presence of a functional cytochrome P450. The COdifference spectrum of microsomal fractions extracted from the emptyvector negative control yeast lack a peak at 450 nm.

Example 6 In Vivo Assay of Recombinant Cytochrome P450s

For functional analysis of these recombinants, one colony is inoculatedinto 5 ml SGI and grown at 30° C. with shaking (250 rpm) forapproximately 24 hours. The cell culture is then transferred to 50 mlfresh SGI, and the cells are grown at 30° C. for 24 hours with shakingat 250 rpm. The cells are collected by centrifugation at 500×g for 5minutes. The cell pellet is resuspended in 20 ml of galactose-containinginduction medium (5 g/L yeast extract, 5 g/L bacto peptone, 20 g/Lgalactose and 1% tergitol Nonidet P40 (Sigma Aldrich. St. Louis, Mo.)).Substrate (3-methyl-5-pentadecatrienyl resorcinol synthesized in-house)is added to a final concentration of 0.2 mM. Cells are allowed tocontinue growing at 28° C. for 16 hours, then are harvested bycentrifugation at 1500×g for 5 minutes. The cell pellet is washed with10 ml of 10 mM K₃PO₄ (potassium phosphate) buffer, pH 7.5. Cells arethen treated for 10 minutes in a Branson ultrasonic water bath (Branson,Danbury, Conn.) with 10 ml methanol. The mixture is clarified bycentrifugation at 1,000×g for 10 minutes. The methanol phase isrecovered and is dried under vacuum. The dried extracts are treated with100 μl of N, N-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in an ovenat 125° C. for 1 hour. The treated extracts then are clarified bycentrifugation at 13,000 rpm for 1 minute, and the clear upper phase isrecovered for GC-MS analysis.

GC-MS analysis is performed with an HP 6890 GC system equipped with aHewlett-Packard HP-5 capillary column (Hewlett Packard, Palo Alto,Calif.) under the following oven conditions: an initial oven temperatureof 120° C. for 2 minutes and a ramp of 20° C./minute to a finaltemperature of 300° C. which is held for 18 minutes. Total run time is29 minutes. One microliter aliquots of BSTFA-derivatized extracts areinjected directly into the gas chromatograph.

Induced cultures of the yeast transformants are incubated with thepredicted substrate, 3-methyl-5-pentadecatrienyl resorcinol, and theproducts are analyzed by GC-MS (FIG. 3). As a control, strains harboringthe empty pYeDP60 vector are cultured in parallel. As seen in FIGS. 3Band 3C, expressing CYP71AM1 or CYP71V7 in yeast with this substrateresults in the appearance of a new peak with a retention time of 14.16minutes and the derivtized parent ion mass of 576 (FIG. 3),corresponding to the calculated molecular mass. When3-methyl-5-pentadecatrienyl resorcinol is incubated with recombinantyeast strains producing sorghum CYP71AM1 or CYP71V7 (FIGS. 3B and 3C),the added substrate with a peak at 13.03 min is metabolized to producethe reduced form of sorgoleone having a peak at 14.16 minutes, which iscomparable to the chromatogram generated from authenticdihydrosorgoleone (FIG. 3A), confirming that CYP71AM1 and CYP71V7 havedihydroxylation activity. Moreover, the ion fragments of the new peakobserved in the mass spectrum are consistent with the fragmentation ofthe dihydrosorgoleone standard (FIG. 4). Taken together, these resultsindicate that both CYP71AM1 and CYP71V7 have pentadecatrienyl resorcinolhydroxylase activity and each encoded enzyme are capable ofhydroxylating 3-methyl-5-pentadecatrienal resorcinol at position 4 and6, resulting in the conversion of the resorcinol into dihydrosorgoleone(a hydroquinone), the direct precursor of sorgoleone (a benzoquinone).The identification and functional characterization of CYP71AM1 andCYP71V7 extends the known range of activities associated with thecytochrome P450 CYP71 family of enzymes.

Example 7 Other Cytochrome P450 Enzyme Isolated Lacking AppropriateEnzymatic Activity

A third EST for which the polynucleotide sequence contained cytochromeP450 domains is isolated according to Example 1. Using the experimentalprotocol set forth in Example 2 but with primers specific for this gene,it is determined that this gene is expressed in both roots and root haircells. Using the protocol set forth in Example 3, but using primersspecific for this gene, a cDNA of the gene is obtained and then issequenced. The DNA sequence of this gene is 49.3% identical to thesequence of SbPRH1 and only 44.1% identical to the sequence of SbPRH2.These percentage identity to SbPRH1 and SbPRH2 are approximately similarto the percentage identity that SbPRH1 shares with SbPRH2. Using theprotocol of Example 4, again with its own set of primers, this gene iscloned into the pYeDP60 expression vector and transfected into S.cerevisiae. P450 functional expression using a carbon monoxidedifferential spectrum at 450 nm per the protocol of Example 5 fails toproduce a peak at 450 nm for this other gene product. Furthermore,in-vivo assays of the recombinant yeast containing the cDNA encodingthis other gene (as set forth in Example 6) fails generate data viaGC-MS that demonstrates that the enzyme encoded by this cDNA metabolizes3-methyl-5-pentadecatrienyl resorcinol to dihydrosorgoleone. While onemay have expected the enzyme encoded by the cDNA would have similaractivity as SbPRH1 and SbPRH2, it is demonstrated that the encodedenzyme does not possess the same activity, even though it containsapproximately the same sequence homology to both SbPRH1 and SbPRH2 asSbPRH1 and SbPRH2 have to each other.

Example 8 Expression of Multiple Sorgoleone Biosynthetic Enzymes inTransgenic Plants

Crops, such as maize (Zea mays), rice (Oryza sativa), soybean (Glycinemax), and others, may be genetically engineered to produce sorgoleone orsorgoleone analogues through the expression of genes associated with thesorgoleone biosynthetic pathway. To generate such transgenic plantsexpressing multiple genes, one uses a binary vector containing multipletransgene expression cassettes, with each cassette containing, at aminimum, a gene promoter, a full-length open reading frame of thetransgene, and a polyadenylation or terminator region. To obtaintransgenic plants capable of producing dihydrosorgoleone, five genes (ortransgenes) are used to transform the plant in question: two fatty aciddesaturases (SbDES2 and SbDES3; U.S. Pat. No. 8,383,890), analkylresorcinol synthase (either SbARS1 or SbARS2; U.S. Patent Pub.2011-0225676), an O-methyltransferase (SbOMT3; U.S. Pat. No. 7,732,666),and either of the two cytochrome P450 enzymes discussed supra, SbPRH1(SEQ ID NO: 11) or SbPRH2 (SEQ ID NO: 13). In the present example, abinary vector system developed by Tzfira, et al., Plant Mol. Biol. 57:503-516 (2005) is used for the expression of an intact sorgoleonebiosynthetic pathway in transgenic plants. However, one can use any ofseveral other vector systems for the assembly of multi-gene expressioncassettes within a binary vector (e.g., Lin, et al., Proc. Natl. Acad.Sci. USA, 100(10), 5962-5967 (2003); Tzfira, et al., 2005; Chung, etal., Trends in Plant Science, 10(8): 357-361 (2005); Schmidt, et al., InVitro Cell.Dev.Biol.-Plant, 44:162-168 (2008)).

The SbARS1 and SbARS2 enzymes possess identical biochemical functions,and similarly, the SbPRH1 and SbPRH2 enzymes possess identicalbiochemical functions, thus only one cDNA encoding each enzyme type isused in the present example. The open reading frames (ORFs) of SbDES2,SbDES3, SbARS1, SbOMT3 and SbPRH1 are first individually PCR-amplifiedusing PfuUltra™ II Fusion HS DNA Polymerase (Agilent Technologies, SantaClara, Calif.), using cDNA clones as template and gene-specific PCRprimers as provided above or in the above cited references and using PCRreaction conditions described above or in the above cited references.The PCR-amplified products are individually subcloned into theintermediate shuttle vector pSAT4 (GeneBank Accession number: DQ005466,Chung, et al. (2005)), generating the following five individualplasmids: 1) pSAT4-DES2 (35S promoter-SbDES2-35S terminator), 2)pSAT4-DES3 (35S promoter-SbDES3-35S terminator), 3) pSAT4-SbARS1 (35Spromoter-SbARS1-35S terminator), 4) pSAT4-SbOMT3 (35Spromoter-SbOMT3-35S terminator), and 5) pSAT4-SbPRH1 (35Spromoter-SbPRH1-35S terminator).

Each expression cassette is then amplified from these intermediateshuttle plasmids by PCR using primer pairs containing recognition sitesfor different rare-cutting restriction endonucleases. In particular, theamplicon generated for the expression cassette containing the 35Spromoter-SbDES2-35S terminator is flanked by recognition sites for therestriction enzyme I-PpoI: the 35S promoter-SbDES3-35S terminator isflanked by recognition sites for I-SceI: the 35S promoter-SbARS1-35Sterminator is flanked by recognition sites for I-CeuI; the 35Spromoter-SbOMT3-35S terminator is flanked by recognition sites forPI-PspI; and the 35S promoter-SbPRH1-35S terminator is flanked byPI-TliI recognition sites. The expression cassettes are then assembledinto the multiple cloning site (MCS) of the Agrobacterium binary vectorpRCS2 (GeneBank Accession number: DQ005454, Chung, et al. (2005)) usingthe existing compatible sites. The PCR amplicons containing the variousexpression cassettes are first digested with the appropriate restrictionenzymes and are then ligated to similarly-treated pRCS2, sequentially,resulting in all of the five cassettes stacked within the T-DNA of asingle binary vector. Prior to stacking these gene cassettes into pRCS2,the vector requires modification in order for the expression of theselectable marker bar gene (coding for phosphinothricin acetyltransferase) to be driven by the CaMV 35S promoter. To accomplish this,the 35S Pro-bar-T35 cassette is first amplified from the plasmid pLH7000(Hausmann and Toepfer, Development of Plasmid Vectors. In Bioengineeringof Custom-Tailored Rape Varieties, D. Brauer, G. Roebbelen, and R.Toepfer, eds (Goettingen, Germany: Gesellschaft fuer Pflanzenzuechtung),pp. 155-171 (1999)) using PCR primers containing recognition sites forthe restriction enzyme AscI. The resulting PCR product is then treatedwith AscI and ligated to the AscI-treated pRCS2 backbone (which includespRCS2 minus the Pocs-bar-Tocs cassette). The final binary vectorcontaining all five expression cassettes is confirmed by DNA sequenceanalysis, and then is mobilized into Agrobacterium lumefaciens strainLBA4404 (but strains EHA 101 or EHA 105 can also be used) for planttransformation.

To express the five sorghum gene products specifically in root haircells of transgenic plants, three published root hair-specific promotersequences, AtEXP7 (Cho and Cosgrove, Plant Cell, 14:3237-3253 (2002)).OsEXPA17 (Yu, et al., The Plant Journal 66:725-734 (2011)) and OsCSLD1(Kim, et al., Plant Physiology, 143:1220-1230 (2007)) are employed. Thepromoter sequences are first amplified from either Arabidopsis (ecotypeCol-0) genomic DNA (AtEXP7 promoter) or rice genomic DNA (cv. Dongjinfor the OsCSLD1 promoter; cv. Kasalath for the OsEXPA17 promoter) usinggene-specific primers. The promoter sequences (AtEXP7 promoter: 2500 bp;OsEXPA17 promoter: 2563 bp; OsCSLD1 promoter: 2500 bp) obtained by PCRare then cloned into the pCR-Blunt II-TOPO vector (Life Technologies,Grand Island, N.Y.) for sequencing to confirm their authenticity. Thesethree promoter sequences are next used to generate the final roothair-specific expression cassettes. The 35S promoter sequences used todirect the expression of each sorghum gene product in the intermediateshuttle vectors (pSAT4 derived vectors) described above are replacedwith root-hair specific promoters using standard cloning procedures. Forexample, the 35S promoter of the 35S promoter-SbDES2-35S terminatorexpression cassette is replaced by the AtEXP7 promoter, resulting in anAtEXP7 promoter-SbDES2-35S terminator cassette with flanking recognitionsites for restriction enzyme I-PpoI. Likewise, the 35S promoter in the35S promoter-SbDES3-35S terminator cassette is replaced by the OsEXPA17promoter to generate an OsEXPA17 promoter-SbDES3-35S terminator cassettewith flanking I-SceI recognition sites. A similar approach is used togenerate an OsCSLD1 promoter-SbARS1-35S terminator cassette withflanking I-CeuI sites, an AtEXP7 promoter-SbOMT3-35S terminator cassettewith flanking PI-PspI sites, and an OsCSLD1 promoter-SbPRH1-35Sterminator cassette with flanking PI-TliI sites. These expressioncassettes are then assembled into the MCS of the modified binary vectorpRCS2 described above. The complete binary vector containing all fivetransgene expression cassettes is mobilized into A. tumefaciens strainLBA4404 (but strains EHA 101 or EHA 105 can also be used) for planttransformation.

Transgenic rice, maize and soybean plants are generated usingAgrobacterium tumefaciens strains harboring the binary vectors describedabove using previously-described methods (for rice: Hiei, et al., ThePlant Journal 6(2):271-282 (1994) and Toki, Plant Molecular BiologyReporter 15:16-211997; for maize: Frame, et al., Plant Physiology129:13-22 (2002); and for soybean: Paz, et al., Plant Cell Reports25:206-213 (2006)). Other plant species are also amenable to theabove-described procedures, or amenable following minor modifications ofthe above procedures.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Alldocuments cited herein are incorporated by reference.

We claim: 1-36. (canceled)
 37. An isolated cDNA comprising a nucleicacid sequence encoding a pentadecatrienyl resorcinol hydroxylase havingthe amino acid sequence of SEQ ID NO:
 14. 38. An isolated cDNAcomprising a nucleic acid sequence of SEQ ID NO:
 13. 39. An expressionvector comprising a promoter and a heterologous polynucleotide; whereinsaid heterologous polynucleotide encodes a pentadecatrienyl resorcinolhydroxylase; wherein said promoter is operatively linked to saidheterologous polynucleotide.
 40. The expression vector of claim 39,wherein said pentadecatrienyl resorcinol hydroxylase has the amino acidsequence of SEQ ID NO:
 14. 41. The expression vector of claim 39,wherein said promoter is an inducible promoter.
 42. The expressionvector of claim 39, wherein said promoter is a constitutive promoter.43. The expression vector of claim 39, wherein said promoter is atissue-specific promoter.
 44. The expression vector of claim 39, whereinsaid heterologous polynucleotide comprises SEQ ID NO:
 13. 45. Theexpression cassette of claim 44 wherein said promoter is an induciblepromoter.
 46. The expression cassette of claim 44 wherein said promoteris a constitutive promoter.
 47. The expression cassette of claim 44wherein said promoter is a tissue-specific promoter.
 48. A transformedcell capable of producing pentadecatrienyl resorcinol hydroxylase, saidtransformed cell comprising the expression vector of claim 39, whereinsaid transformed cell is selected from the group consisting of a plantcell, a fungus, a blue-green algae, and a bacterium.
 49. The transformedcell of claim 48, wherein said pentadecatrienyl resorcinol hydroxylasehas the amino acid sequence of SEQ ID NO:
 14. 50. The transformed cellof claim 48, wherein said heterologous polynucleotide comprises SEQ IDNO:
 13. 51. A transgenic organism capable of producing pentadecatrienylresorcinol hydroxylase, said transgenic organism comprising thetransformed cell of claim 48; wherein said transgenic organism produceselevated levels of pentadecatrienyl resorcinol hydroxylase compared tothe levels of pentadecatrienyl resorcinol hydroxylase produced by thenon-transformed organism.
 52. The transgenic organism of claim 51,wherein said pentadecatrienyl resorcinol hydroxylase has the amino acidsequence of SEQ ID NO:
 14. 53. The transgenic organism of claim 51,wherein said heterologous polynucleotide comprises SEQ ID NO:
 13. 54. Atransformed cell that has elevated levels of pentadecatrienyl resorcinolhydroxylase compared to the level of pentadecatrienyl resorcinolhydroxylase present in the untransformed cell; said transformed cellcomprises an expression vector, wherein said expression vector comprisesa promoter operably linked to heterologous DNA encoding saidpentadecatrienyl resorcinol hydroxylase, and wherein saidpentadecatrienyl resorcinol hydroxylase has the amino acid sequence ofSEQ ID NO:
 14. 55. A method of manipulating pentadecatrienyl resorcinolhydroxylase levels in a transformed cell or transformed organism, saidmethod comprising introducing into a wild-type cell or a wild-typeorganism an expression vector comprising a heterologous polynucleotideand a promoter to produce said transformed cell or said transformedorganism, wherein said heterologous polynucleotide is operably linked tosaid promoter, wherein said promoter is active in said cell or organism,wherein said heterologous polynucleotide encodes a polypeptide havingthe amino acid sequence of SEQ ID NO: 14, wherein said polypeptide haspentadecatrienyl resorcinol hydroxylase activity, wherein saidtransformed cell or transformed organism is selected from the groupconsisting of a plant, a fungus, a blue-green algae, and a bacterium,and allowing the production of pentadecatrienyl resorcinol hydroxylasein said transformed cell or transformed organism.
 56. A transformedplant cell or plant made by the method of claim 55, or progeny thereof,wherein said transformed plant cell or plant or progeny thereof isselected for having increased levels of pentadecatrienyl resorcinolhydroxylase as compared to the level of pentadecatrienyl resorcinolhydroxylase present in the non-transformed plant cell or plant of thesame variety.